Biodistribution and function of extracellular miRNA-155 in mice

Abstract

Circulating miRNAs can be found in extracellular vesicles (EV) and could be involved in intercellular communication. Here, we report the biodistribution of EV associated miR-155 using miR-155 KO mouse model. Administration of exosomes loaded with synthetic miR-155 mimic into miR-155 KO mice resulted in a rapid accumulation and clearance of miR-155 in the plasma with subsequent distribution in the liver, adipose tissue, lung, muscle and kidney (highest to lowest, respectively). miR-155 expression was detected in isolated hepatocytes and liver mononuclear cells of recipient KO mice suggesting its cellular uptake. In vitro, exosome-mediated restoration of miR-155 in Kupffer cells from miR-155 deficient mice augmented their LPS-induced MCP1 mRNA increase. The systemic delivery of wild type plasma to miR-155 KO mice also resulted in a rapid accumulation of miR-155 in the circulation and distribution to the liver and adipose tissue. In summary, our results demonstrate tissue biodistribution and biologic function of EV-associated miR-155.

Figure 1. Biodistribution of exosome loaded miRNA-155 mimic in miRNA-155 KO mice.

C57Bl/6 wild type female mice were either injected with saline or 2.5 mg/kg CpG DNA (i.p.) once a day for three days and on day 4 CpG treated mice received 0.5 mg/kg LPS for 3h and blood was isolated. Extracellular vesicles were isolated from plasma using filtration and ExoQuick and miR-155 levels were determined (A) B cells were treated with CD40 and IL-4 and exosomes were isolated using CD63 magnetic beads as described in the methods. Exosomes were electroporated with either scrambled mimic or miRNA-155 mimic and miR-155 levels were determined after RNase A treatment. miR-155 levels were normalized to scrambled mimic loaded exosomes (B) 100 ul exosomes (loaded with scrambled mimic or miR-155 mimic) were injected (i.v) into miR-155 KO mice for indicated times and mice were perfused as described in the methods. miR-155 levels in the plasma (C) liver (D) adipose tissue (E) lung (F) muscle (G) kidney (H) isolated hepatocytes (I) and mononuclear cells (J) was determined using real time qPCR. miR-155 levels were normalized to wild type saline treated mice. * indicates p < 0.05 versus scrambled mimic exosome treated KO mice. Synthetic spiked cel-miR-39 (A–C) or SnoRNA202 (D–J) was used to normalize the technical variations between the samples. Statistical analysis was performed using non-parametric Mann-Whitney test.

Figure 2. Biological function of exosome loaded miRNA-155 mimic.

Hepatocytes and Kupffer cells were isolated from miR-155 KO mice as described in the methods. Exosomes loaded with either scrambled mimic or miR-155 mimic were added to cells for 6 h. Cells were washed 3 times with PBS to remove free-floating exosomes. Hepatocytes were lysed with Qiazole and miR-155 levels were evaluated (A) Kupffer cells were treated or not with LPS (100 ng/ul) for 6 h and cell-free supernatant was collected. The levels of miR-155 (B) MIP2 (C) and MCP1 (mRNA) were checked using real-time qPCR and levels were normalized to untreated cells. MCP1 protein levels were measured from cell-free supernatant (E) miR-155 levels were normalized to hepatocytes or Kupffer cells isolated from wild type mice. * indicates p<0.05 scrambled loaded exosome treated cells and # compared to scrambled loaded exosome +LPS treated cells. SnoRNA202 (A,B) or 18S (C,D) was used to normalize the technical variations between the samples. ns: non significant. Statistical analysis was performed using non-parametric Mann-Whitney test.

Figure 3. Biodistribution of transferred wild type plasma in miRNA-155 KO mice.

miR-155 KO mice (n = 6–8) were injected with ~150 ul of plasma isolated from WT mice (CpG+LPS treated) as described in the methods. After systemic administration, blood was collected and mice were perfused or not as indicated. Plasma was isolated and miR-155 levels were determined by real-time qPCR. Due to miR-155 low signal, pre-amplification of cDNA from tissue samples was performed. i.v route of delivery (A) and i.p route of delivery (B) miR-155 levels in non-perfused (C) and perfused livers (D) miR-155 levels were normalized to wild type saline treated mice. Data is presented as mean ± SEM. * indicates p < 0.05 versus saline treated KO mice. nd: not detected. Synthetic spiked cel-miR-39 (A,B) or SnoRNA202 (C,D) was used to normalize the technical variations between the samples. Statistical analysis was performed using non-parametric Mann-Whitney test.

Figure 4. Summary of miR-155 biodistribution.